MSCV LTRmir30 vector sequencing! - (Feb/24/2007 )
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Hey!
I'm trying to sequence MSCV vector, before moving further with the virus work. I made 14!!!!!! different constructs, two different MSCV vectors with inserted linkers. The size of each linker is 110 bp, ( Hanon's lab protocol). I spent ages picking colonies, making mini-preps. Then I checked them by restriction enzymes, two diffrent sets of enzymes. Everything looked perfect. I was getting or 110 bp size fragment, or was looking for the difference 110bp. Anyway then I made midi-preps on selected preps and wanted sequence linkers. And what!!!!!!!! Nothing, I'm using LMP primer, but or there are no picks, or many NNN in my sequencing. I'm so stressed and worried, I have to finished vectors, and make the virus titer as soon as possible. But what you think what to do now. I have tried another kit for sequencing but on midi-preps the same bad results. Today I prepered sequencing on mini-preps but maybe there is too little DNA. Maybe I should run all vectors on some GFX columns or something to purify it more. Please, help I'm getting crazy with that. I already prepered everything in virus lab, based on restriction enzymes results, I do have those linkers inside, but since they have mir30 and very strong secendary structure I cant sequenced them. OHHHHH
MJL:(
Hey!
I'm trying to sequence MSCV vector, before moving further with the virus work. I made 14!!!!!! different constructs, two different MSCV vectors with inserted linkers. The size of each linker is 110 bp, ( Hanon's lab protocol). I spent ages picking colonies, making mini-preps. Then I checked them by restriction enzymes, two diffrent sets of enzymes. Everything looked perfect. I was getting or 110 bp size fragment, or was looking for the difference 110bp. Anyway then I made midi-preps on selected preps and wanted sequence linkers. And what!!!!!!!! Nothing, I'm using LMP primer, but or there are no picks, or many NNN in my sequencing. I'm so stressed and worried, I have to finished vectors, and make the virus titer as soon as possible. But what you think what to do now. I have tried another kit for sequencing but on midi-preps the same bad results. Today I prepered sequencing on mini-preps but maybe there is too little DNA. Maybe I should run all vectors on some GFX columns or something to purify it more. Please, help I'm getting crazy with that. I already prepered everything in virus lab, based on restriction enzymes results, I do have those linkers inside, but since they have mir30 and very strong secendary structure I cant sequenced them. OHHHHH
MJL:(
Because of secondary structures it is difficult to sequence viral plasmids. You might have to use DMSO when you amplify for sequencing. Better ask the sequencing lab, explaining the problem to give some suggestions.
Hey!
I'm trying to sequence MSCV vector, before moving further with the virus work. I made 14!!!!!! different constructs, two different MSCV vectors with inserted linkers. The size of each linker is 110 bp, ( Hanon's lab protocol). I spent ages picking colonies, making mini-preps. Then I checked them by restriction enzymes, two diffrent sets of enzymes. Everything looked perfect. I was getting or 110 bp size fragment, or was looking for the difference 110bp. Anyway then I made midi-preps on selected preps and wanted sequence linkers. And what!!!!!!!! Nothing, I'm using LMP primer, but or there are no picks, or many NNN in my sequencing. I'm so stressed and worried, I have to finished vectors, and make the virus titer as soon as possible. But what you think what to do now. I have tried another kit for sequencing but on midi-preps the same bad results. Today I prepered sequencing on mini-preps but maybe there is too little DNA. Maybe I should run all vectors on some GFX columns or something to purify it more. Please, help I'm getting crazy with that. I already prepered everything in virus lab, based on restriction enzymes results, I do have those linkers inside, but since they have mir30 and very strong secendary structure I cant sequenced them. OHHHHH
MJL:(
Because of secondary structures it is difficult to sequence viral plasmids. You might have to use DMSO when you amplify for sequencing. Better ask the sequencing lab, explaining the problem to give some suggestions.
Thank you, will do that. Anyway,people who used that vector told me that the sequencing is the standard procedure....
